Product Class: Other

T4 DNA Ligase
NEBU recombinant 16 65 Heat

Looking for T4 DNA Ligase alternatives?  Try Salt-T4 or Hi-T4 DNA Ligases

Product Introduction

T4 DNA Ligase will ligate these substrates:

dsDNA




Nicked DNA/RNA





 

Catalog # Size Concentration
M0202S 20000.0 units 400000 units/ml
M0202T 20000.0 units 2000000 units/ml
M0202L 100000.0 units 400000 units/ml
M0202M 100000.0 units 2000000 units/ml

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    Quick Tip - What is the ideal incubation temperature for ligation?

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    Quick Tip - When is a long incubation time necessary for ligation?

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Product Information

Description

Highlights

  • Isolated from a recombinant source
  • Supplied with 10X Reaction Buffer
For details on NEB's quality controls for DNA ligases, visit our Ligase Quality page.

T4 DNA Ligase Competitor Study - Nuclease Contamination
T4 DNA Ligase Competitor Study- Nuclease Contamination
T4 DNA Ligase from multiple suppliers was tested in reactions containing a fluorescent labeled single stranded, double stranded blunt, 3’overhang or 5’ overhang containing oligonucleotides. The percent degradation by contaminating nucleases is determined by capillary electrophoresis and peak analysis. The resolution is at the single nucleotide level.

Product Source

An E. coli strain that carries the cloned T4 DNA Ligase gene.
This product is related to the following categories:
DNA Ligases Products
This product can be used in the following applications:
BioBrick® Assembly,
Cloning Ligation,
NEBridge® Golden Gate Assembly

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Unit Definition

One unit is defined as the amount of enzyme required to give 50% ligation of HindIII fragments of λ DNA (5´ DNA termini concentration of 0.12 µM, 300- µg/ml) in a total reaction volume of 20 μl in 30 minutes at 16°C in 1X T4 DNA Ligase Reaction Buffer.

Concentration:
400,000 cohesive end units/ml and 2,000,000 cohesive end units/ml

Reaction Conditions

1X T4 DNA Ligase Reaction Buffer
Incubate at 16°C

1X T4 DNA Ligase Reaction Buffer
50 mM Tris-HCl
10 mM MgCl2
1 mM ATP
10 mM DTT
(pH 7.5 @ 25°C)

Storage Buffer

10 mM Tris-HCl
50 mM KCl
1 mM DTT
0.1 mM EDTA
50% Glycerol
pH 7.4 @ 25°C

Heat Inactivation

65°C for 10 minutes

Application Features

  • Cloning of restriction fragments
  • Joining linkers and adapters to blunt-ended DNA

Product Notes

  1. ATP is an essential cofactor for the reaction. This contrasts with E. coli DNA ligase which requires NAD.
  2. To dilute T4 DNA Ligase that will subsequently be stored at -20°C, 50% glycerol storage buffer (Diluent Buffer A (with rAlbumin), NEB #B8532S) should be used; to dilute for immediate use, 1X T4 DNA Ligase Reaction Buffer can be used.
  3. Ligation can also be performed in any of the four restriction endonuclease NEBuffers or in T4 Polynucleotide Kinase Buffer if they are supplemented with 1 mM ATP.
  4. Room Temperature Ligation
    For convenience, ligations may be done at room temperature (20-25°C). For cohesive (sticky) ends, use 1 µl of T4 DNA Ligase in a 20 µl reaction for 10 minutes. For blunt ends, use 1 µl of T4 DNA Ligase in a 20 µl reaction for 2 hours or 1 µl high concentration T4 DNA Ligase for 10 minutes. Alternatively, NEB's Quick Ligation Kit (#M2200S) [30 reactions] or (#M2200L) [150 reactions]) is uniquely formulated to ligate both blunt and cohesive (sticky) ends in 5 minutes at room temperature.

References

  1. Engler, M.J. and Richardson, C.C. (1982). P.D. Boyer(Ed.), 5, 3. San Diego: Academic Press.
  2. Remaut, E., Tsao, H. and Fiers, W. (1983). Gene. 22, 103-113.
  3. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, (2nd Ed.). 1.53-1.73.

Protocols, Manuals & Usage

Protocols

  1. Ligation Protocol with T4 DNA Ligase (M0202)
  2. Golden Gate (24 Fragment) Assembly Protocol

Usage & Guidelines

Application Notes

Tools & Resources

Selection Charts

Web Tools

FAQs & Troubleshooting

FAQs

  1. What are some potential problems with the ligation reaction using T4 DNA Ligase that can lead to transformation failure?
  2. What are some other problems that should be considered when trouble shooting a transformation problem?
  3. What problems can be encountered in the restriction digest that can cause ligation using T4 DNA Ligase or subsequent transformation to fail?
  4. What controls should be run to test the cells and DNA when using T4 DNA Ligase?
  5. When should T4 DNA Ligase be the enzyme of choice?
  6. Can the T4 DNA Ligase be used with the Quick Ligase buffer?
  7. What is the definition of a Weiss Unit and a Cohesive End Unit?
  8. What is the difference between the two definitions and why does NEB use the Cohesive End Unit?
  9. How much DNA should be used in a ligation using T4 DNA Ligase?
  10. Can T4 DNA Ligase be used in other NEBuffers, including rCutSmart?
  11. Can T4 DNA Ligase be heat inactivated?

Troubleshooting

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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